Polypeptides having arabinofuranosidase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having alpha-L-arabinofuranosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/991,484, filed Nov. 30, 2007, which application is incorporatedherein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingalpha-L-arabinofuranosidase activity and isolated polynucleotidesencoding the polypeptides. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing and using the polypeptides.

2. Description of the Related Art

Plant cell wall polysaccharides generally constitute 90% of the plantcell wall and can be divided into three groups: cellulose,hemicellulose, and pectin. Cellulose represents the major constituent ofcell wall polysaccharides. Hemicelluloses are the second most abundantconstituent of plant cell walls. The major hemicellulose polymer isxylan. The structure of xylans found in cell walls of plants can differsignificantly depending on their origin, but they always contain abeta-1,4-linked D-xylose backbone. The beta-1,4-linked D-xylose backbonecan be substituted by various side groups, such as L-aribinose,D-galactose, acetyl, feruloyl, p-coumaroyl, and glucuronic acidresidues.

Biodegradation of the xylan backbone depends on two classes of enzymes:endoxylanases and beta-xylosidases. Endoxylanases (EC 3.2.1.8) cleavethe xylan backbone into smaller oligosaccharides, which can be furtherdegraded to xylose by beta-xylosidases (EC 3.2.1.37). Other enzymesinvolved in the degradation of xylan include, for example, acetylxylanesterase, arabinase, alpha-L-arabinofuranosidase, alpha-glucuronidase,feruloyl esterase, and p-coumaric acid esterase.

Kaji and Tagawa, 1970, Biochim. Biophys. Acta 207 456-464, describe thepurification, crystallization and amino acid composition of analpha-L-arabinofuranosidase from Aspergillus niger. Kaji. and Yoshihara,1971, Biochim. Biophys. Acta 250 367-371, describe the properties of analpha-L-arabinofuranosidase from Corticium rolfsii. Tagawa and Kaji,1969, Carbohydr. Res. 11: 293-301, describe the preparation ofL-arabinose-containing polysaccharides and the action of analpha-L-arabinofuranosidase on the polysaccharides. Filho et al., 1996,Appl. Environ. Microbiol. 62: 168-173, disclose the purification andcharacterization of two alpha-L-arabinofuranosidases from Penicilliumcapsulatum. WO 96/06935 discloses an arabinoxylan degrading enzyme fromAspergillus niger. WO 2006/125438 describes a Penicillium capsulatumalpha-L-arabinofuranosidase and polynucleotide thereof.

The present invention relates to polypeptides havingalpha-L-arabinofuranosidase activity and polynucleotides encoding thepolypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingalpha-L-arabinofuranosidase activity selected from the group consistingof:

(a) a polypeptide comprising an amino acid sequence having at least 70%sequence identity to the mature polypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast medium-high stringency conditions with the mature polypeptidecoding sequence of SEQ ID NO: 1 or its full-length complementary strand;

(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 70% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having alpha-L-arabinofuranosidase activity, selected fromthe group consisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence having at least 70% sequence identity to the mature polypeptideof SEQ ID NO: 2;

(b) a polynucleotide that hybridizes under at least medium-highstringency conditions with the mature polypeptide coding sequence of SEQID NO: 1 or its full-length complementary strand;

(c) a polynucleotide comprising a nucleotide sequence having at least70% sequence identity to the mature polypeptide coding sequence of SEQID NO: 1; and

(d) a polynucleotide encoding a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NO: 2.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing a polypeptide havingalpha-L-arabinofuranosidase activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide in a cell, comprising administering to thecell or expressing in the cell a double-stranded RNA (dsRNA) molecule,wherein the dsRNA comprises a subsequence of a polynucleotide of thepresent invention. The present also relates to such a double-strandedinhibitory RNA (dsRNA) molecule, wherein optionally the dsRNA is a siRNAor a miRNA molecule.

The present invention also relates to methods for degrading a materialcomprising a xylan.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding such a polypeptide havingalpha-L-arabinofuranosidase activity.

The present invention also relates to methods of producing such apolypeptide having alpha-L-arabinofuranosidase, comprising: (a)cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding such a polypeptide havingalpha-L-arabinofuranosidase activity under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide comprising orconsisting of amino acids 1 to 17 of SEQ ID NO: 2, wherein the gene isforeign to the nucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence and the deduced amino acidsequence of a Family 62 Humicola insolens DSM 1800alpha-L-arabinofuranosidase (SEQ ID NOs: 1 and 2, respectively).

FIG. 2 shows a restriction map of pMMar4.

FIG. 3 shows a restriction map of pHinsGH62A.

DEFINITIONS

Alpha-L-arabinofuranosidase activity: The term“alpha-L-arabinofuranosidase activity” is defined herein as analpha-L-arabinofuranoside arabinofuranohydrolase activity (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeactivity acts on alpha-L-arabinofuranosides, alpha-L-arabinanscontaining (1,3)- and/or (1,5)-linkages, arabinoxylans, andarabinogalactans. Alpha-L-arabinofuranosidase is also known asarabinosidase, alpha-arabinosidase, alpha-L-arabinosidase,alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase,alpha-L-arabinofuranoside hydrolase, L-arabinosidase, oralpha-L-arabinanase. For purposes of the present invention,alpha-L-arabinofuranosidase activity is determined according to theprocedures described below or the procedure described in Example 10. Thepolypeptides of the present invention have at least 20%, preferably atleast 40%, more preferably at least 50%, more preferably at least 60%,more preferably at least 70%, more preferably at least 80%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 100% of the alpha-L-arabindfuranosidase activity ofthe mature polypeptide of SEQ ID NO: 2.

Preparation of specific arabinoxylan oligosaccharides. Oligosaccharidescontaining arabinosyl groups linked terminally (1→3) are prepared byincubating 1 g of water insoluble wheat arabinoxylan (Megazyme, Bray,County Wicklow, Ireland) in 100 ml of 0.1 M acetate pH 6.0 buffer with6.67 g of SHEARZYME™ (Aspergillus aculeatus GH10 endo-1,4-beta-xylanase,Novozymes A/S, Bagsvaerd, Denmark) per kg of water insoluble wheatarabinoxylan for 2 hours at 30° C. Oligosaccharides containingarabinosyl groups linked internally (1→3) are prepared by incubating 1 gof water insoluble wheat arabinoxylan in 100 ml of 0.1 M sodium acetatepH 6.0 with 0.03 g of PENTOPAN™ MONO (Thermomyces lanuginosus GH11endo-1,4-β-xylanase; Novozytnes A/S, Bagsvaerd, Denmark) per kg of waterinsoluble wheat arabinoxylan for 2 hours at 30° C. Oligosaccharidescontaining arabinosyl groups linked internally (1→2) are prepared byincubating 1 g of water insoluble wheat arabinoxylan in 100 ml of 0.1 Msodium acetate pH 6.0 with 0.03 g of PENTOPAN™ MONO per kg of waterinsoluble wheat arabinoxylan and alpha-L-arabinofuranosidase per kg ofwater soluble wheat arabinoxylan for 2 hours at 30° C. To stop theenzymatic reaction, the mixture is heated at 100° C. for 10 minutes. Thearabinoxylo-oligosaccharides are concentrated on a rotary evaporator andevaluated by ¹H-NMR.

Determination of optimal reaction conditions. The optimal reactionconditions for an alpha-L-arabinofuranosidase is evaluated in atwo-factor Box-Behnken response surface design templates (Montgomery,2001, Design and analysis of experiments. Wiley, New York). Eachtemplate comprises several different combinations of pH (3-7) andreaction temperature (30-70° C.) with 3 center points. Water solublewheat arabinoxylan (0.002 g; Megazyme, Bray, County Wicklow, Ireland) isdissolved in 2 ml of deionized water. The solution is then incubatedwith 0.1 g of enzyme protein per kg of water soluble wheat arabinoxylanper assay. Samples are withdrawn after exactly 24 hours of reaction andheated immediately at 100° C. for 10 minutes to stop the enzymereaction. The samples are then centrifuged at 20,000×g for 10 minutesand the level of arabinose is determined in the supernatants by HPAECanalysis. The values reported are in mg per g of wheat arabinoxylan.

Mode of action of alpha-L-arabinofuranosidases.Alpha-L-arabinofuranosidase is added to 0.01 g of water soluble wheatarabinoxylan, 0.01 g of oligosaccharides containing either arabinosylgroups linked terminally (1→3), 0.01 g of oligosaccharides containingarabinosyl groups linked internally (1→3), or 0.01 g of oligosaccharidescontaining arabinosyl groups linked internally (1→2) in 1 ml of 0.1 Msodium acetate pH 6.0 for 2 hours at 40° C. The enzymatic reactions areinactivated at 100° C. for 10 minutes. Samples are concentrated on arotary evaporator and analysed by ¹H-NMR.

HPAEC. Hydrolysates (10 μl) are applied onto a BioLC system (DionexCorporation, Sunnyvale, Calif., USA) fitted with a CARBOPAC™ PA1 guardcolumn (4×250 mm) (Dionex Corporation, Sunnyvale, Calif., USA) combinedwith a CARBOPAC™ PA1 precolumn (4×50 mm). Arabinose is separatedisocratically with 10 mM KOH for 15 minutes at a flow-rate of 1 ml perminute. Arabinose is detected by a pulsed electrochemical detector inthe pulsed amperiometric detection mode. The potential of the electrodeis programmed for +0.1 V (t=0-0.4 s) to −2.0 V (t=0.41-0.42 s) to 0.6 V(t=0.43 s) and finally −0.1 V (t=0.44-0.50 s), while integrating theresulting signal from t=0.2-0.4 s. Arabinose (Merck, Darmstadt, Germany)is used as a standard.

¹H-NMR analysis. All degradation products are lyophilized twice from99.9% D₂O and re-dissolved in 99.9% D₂O. Some hydrolysates are dialyzed(Spectra/Por membrane molecular weight cut-off 1000) to remove freearabinose prior to the spectral analysis. The ¹H-NMR spectra arerecorded at 30° C. in a Varian Mercury-VX instrument operated at 400 MHzand equipped with a 4-nucleus auto-switchable probe. Data are collectedover 128-512 scans and the HDO signal is used as a reference signal(4.67 ppm).

Xylan-containing material: The term “xylan-containing material” isdefined herein as any material comprising xylan as a constituent. Xylanis a plant cell wall polysaccharide containing a backbone ofbeta-1,4-linked xylose residues. Side chains of 4-O-methylglucuronicacid and arabinose are generally present in varying amounts, togetherwith acetyl and feruloyl groups. Xylan is a major constituent ofhemicellulose.

Family 62 or Family GH62: The term “Family 62” or “Family GH62” or“GH62” is defined herein as a polypeptide falling into the glycosidehydrolase Family 62 according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form, i.e.,that the polypeptide preparation is essentially free of otherpolypeptide material with which it is natively or recombinantlyassociated. This can be accomplished, for example, by preparing thepolypeptide by well-known recombinant methods or by classicalpurification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having alpha-L-arabinofuranosidase activity that is in itsfinal form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In a preferred aspect, the maturepolypeptide is amino acids 18 to 387 of SEQ ID NO: 2 based on theSignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 2 are asignal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having alpha-L-arabinofuranosidase activity. In apreferred aspect, the mature polypeptide coding sequence is nucleotides52 to 1161 of SEQ ID NO: 1 based on the SignalP program that predictsnucleotides 1 to 51 of SEQ ID NO: 1 encode a signal peptide.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice of al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein that gives an E value (or expectancy score) of lessthan 0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Humicola insolens alpha-L-arabinofuranosidase of SEQ ID NO: 2or the mature polypeptide thereof.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of the mature polypeptide of SEQ IDNO: 2; or a homologous sequence thereof; wherein the fragment hasalpha-L-arabinofuranosidase activity. In a preferred aspect, a fragmentcontains at least 310 amino acid residues, more preferably at least 330amino acid residues, and most preferably at least 350 amino acidresidues of the mature polypeptide of SEQ ID NO: 2 or a homologoussequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of the mature polypeptide coding sequence of SEQ ID NO: 1;or a homologous sequence thereof; wherein the subsequence encodes apolypeptide fragment having alpha-L-arabinofuranosidase activity. In apreferred aspect, a subsequence contains at least 930 nucleotides, morepreferably at least 990 nucleotides, and most preferably at least 1050nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 ora homologous sequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99%, and even most preferably at least 99.5%pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2; or a homologous sequence thereof; as well as geneticmanipulation of the DNA encoding such a polypeptide. The modificationcan be a substitution, a deletion and/or an insertion of one or more(several) amino acids as well as replacements of one or more (several)amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having alpha-L-arabinofuranosidase activity producedby an organism expressing a modified polynucleotide sequence of themature polypeptide coding sequence of SEQ ID NO: 1; or a homologoussequence thereof. The modified nucleotide sequence is obtained throughhuman intervention by modification of the polynucleotide sequencedisclosed in SEQ ID NO: 1; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides HavingAlpha-L-Arabinofuranosidase Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising an amino acid sequence having a degree ofsequence identity to the mature polypeptide of SEQ ID NO: 2 ofpreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which havealpha-L-arabinofuranosidase activity (hereinafter “homologouspolypeptides”). In a preferred aspect, the homologous polypeptides havean amino acid sequence that differs by ten amino acids, preferably byfive amino acids, more preferably by four amino acids, even morepreferably by three amino acids, most preferably by two amino acids, andeven most preferably by one amino acid from the mature polypeptide ofSEQ ID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof having alpha-L-arabinofuranosidase activity. In apreferred aspect, the polypeptide comprises the amino acid sequence ofSEQ ID NO: 2. In another preferred aspect, the polypeptide comprises themature polypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 18 to 387 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof havingalpha-L-arabinofuranosidase activity. In another preferred aspect, thepolypeptide comprises amino acids 18 to 387 of SEQ ID NO: 2. In anotherpreferred aspect, the polypeptide consists of the amino acid sequence ofSEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof havingalpha-L-arabinofuranosidase activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of amino acids 18 to 387 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof havingalpha-L-arabinofuranosidase activity. In another preferred aspect, thepolypeptide consists of amino acids 18 to 387 of SEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having alpha-L-arabinofuranosidase activity that areencoded by polynucleotides that hybridize under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (I) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) a subsequence of (i),or (iii) a full-length complementary strand of (i) or (ii) (J. Sambrook,E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.). A subsequence of themature polypeptide coding sequence of SEQ ID NO: 1 contains at least 100contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmenthaving alpha-L-arabinofuranosidase activity. In a preferred aspect, thecomplementary strand is the full-length complementary strand of themature polypeptide coding sequence of SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; aswell as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof;may be used to design nucleic acid probes to identify and clone DNAencoding polypeptides having alpha-L-arabinofuranosidase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 14, preferably at least25, more preferably at least 35, and most preferably at least 70nucleotides in length. It is, however, preferred that the nucleic acidprobe is at least 100 nucleotides in length. For example, the nucleicacid probe may be at least 200 nucleotides, preferably at least 300nucleotides, more preferably at least 400 nucleotides, or mostpreferably at least 500 nucleotides in length. Even longer probes may beused, e.g., nucleic acid probes that are preferably at least 600nucleotides, more preferably at least 700 nucleotides, even morepreferably at least 800 nucleotides, or most preferably at least 900nucleotides in length. Both DNA and RNA probes can be used. The probesare typically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having alpha-L-arabinofuranosidaseactivity. Genomic or other DNA from such other strains may be separatedby agarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that is homologouswith SEQ ID NO: 1; or a subsequence thereof; the carrier material ispreferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1;its full-length complementary strand; or a subsequence thereof; undervery low to very high stringency conditions. Molecules to which thenucleic acid probe hybridizes under these conditions can be detectedusing, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 52 to 1161 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencethat encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pHinsGH62A which is contained in E. coliNRRL B-50075, wherein the polynucleotide sequence thereof encodes apolypeptide having alpha-L-arabinofuranosidase activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding region contained in plasmid pHinsGH62A which is contained in E.coli NRRL B-50075.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having alpha-L-arabinofuranosidase activity encoded bypolynucleotides comprising or consisting of nucleotide sequences thathave a degree of sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 of preferably at least 70%, more preferably atleast 75%, more preferably at least 80%, more preferably at least 85%,even more preferably at least 90%, most preferably at least 95%, andeven most preferably at least 96%, at least 97%, at least 98%, or atleast 99%, which encode an active polypeptide. See polynucleotidesection herein.

In a fourth aspect, the present invention relates to artificial variantscomprising a substitution, deletion, and/or insertion of one or more (orseveral) amino acids of the mature polypeptide of SEQ ID NO: 2; or ahomologous sequence thereof. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification, bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,alpha-L-arabinofuranosidase activity) to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton ofal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett 309: 59-64. The identities of essential amino acidscan also be inferred from analysis of identities with polypeptides thatare related to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, such as aminoacids 18 to 387 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

Sources of Polypeptides Having Alpha-L-Arabinofuranosidase Activity

A polypeptide having alpha-L-arabinofuranosidase activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a nucleotide sequence is produced by the source or by a strain inwhich the nucleotide sequence from the source has been inserted. In apreferred aspect, the polypeptide obtained from a given source issecreted extracellularly.

A polypeptide having alpha-L-arabinofuranosidase activity of the presentinvention may be a bacterial polypeptide. For example, the polypeptidemay be a gram positive bacterial polypeptide such as a Bacillus,Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacilluspolypeptide having alpha-L-arabinofuranosidase activity, or a Gramnegative bacterial polypeptide such as an E. coli, Pseudomonas,Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,Ilyobacter, Neisseria, or Ureaplasma polypeptide havingalpha-L-arabinofuranosidase activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having alpha-L-arabinofuranosidase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide havingalpha-L-arabinofuranosidase activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingalpha-L-arabinofuranosidase activity.

A polypeptide having alpha-L-arabinofuranosidase activity of the presentinvention may also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kiuyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide havingalpha-L-arabinofuranosidase activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Mycellophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide havingalpha-L-arabinofuranosidase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide havingalpha-L-arabinofuranosidase activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Irpex lacteus, Mucormiehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumfuniculosum, Penicillium purpurogenum, Phanerochaete chrysosporium,Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa,Thielavia australeinsis, Thielavia fimeti, Thielavia microspore,Thielavia ovispora, Thielavia peruviana, Thielavia spededonium,Thielavia setosa, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptidehaving alpha-L-arabinofuranosidase activity.

In another preferred aspect, the polypeptide is a Humicola grisea,Humicola insolens, or Humicola lanuginosa polypeptide.

In a more preferred aspect, the polypeptide is a Humicola insolenspolypeptide having alpha-L-arabinofuranosidase activity. In a mostpreferred aspect, the polypeptide is a Humicola insolens DSM 1800polypeptide having alpha-L-arabinofuranosidase activity, e.g., thepolypeptide comprising the mature polypeptide of SEQ ID NO: 2.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having alpha-L-arabinofuranosidase activity from the fusionprotein. Examples of cleavage sites include, but are not limited to, aKex2 site that encodes the dipeptide Lys-Arg (Martin et al., 2003, J.Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J.Biotechnol. 76; 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ.Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503;and Contreras at al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton at al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie at al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter at al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that encodepolypeptides having alpha-L-arabinofuranosidase activity of the presentinvention.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pHinsGH62Awhich is contained in E. coli NRRL B-50075. In another preferred aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 52 to 1161 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in plasmid pHinsGH62A which is contained in E, coli NRRLB-50075. The present invention also encompasses nucleotide sequencesthat encode polypeptides comprising or consisting of the amino acidsequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differfrom SEQ ID NO: 1 or the mature polypeptide coding sequence thereof byvirtue of the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ IDNO: 2 that have alpha-L-arabinofuranosidase activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 1, in which the mutant nucleotide sequenceencodes the mature polypeptide of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Humicola, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofsequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the mature polypeptide coding sequenceof SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested foralpha-L-arabinofuranosidase activity to identify amino acid residuesthat are critical to the activity of the molecule. Sites ofsubstrate-enzyme interaction can also be determined by analysis of thethree-dimensional structure as determined by such techniques as nuclearmagnetic resonance analysis, crystallography or photoaffinity labeling(see, e.g., de Vos et al., 1992, supra; Smith et al., 1992, supra;Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 1 or its full-lengthcomplementary strand; or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 1 or its full-lengthcomplementary strand; and (b) isolating the hybridizing polynucleotide,which encodes a polypeptide having alpha-L-arabinofuranosidase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide, The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae those phosphate isomerase);and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding sequence that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingsequence that is foreign to the coding sequence. The foreign signalpeptide coding sequence may be required where the coding sequence doesnot naturally contain a signal peptide coding sequence. Alternatively,the foreign signal peptide coding sequence may simply replace thenatural signal peptide coding sequence in order to enhance secretion ofthe polypeptide. However, any signal peptide coding sequence thatdirects the expressed polypeptide into the secretory pathway of a hostcell of choice, i.e., secreted into a culture medium, may be used in thepresent invention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 17 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding sequence comprises or consists of nucleotides 1 to 51 ofSEQ ID NO: 1.

The control sequence may also be a propeptide coding sequence that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propeptide is generallyinactive and can be converted to a mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding sequence may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors.

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the nucleotide sequence encoding the polypeptide at suchsites. Alternatively, a polynucleotide sequence of the present inventionmay be expressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of sequence identity to thecorresponding target sequence to enhance the probability of homologousrecombination. The integrational elements may be any sequence that ishomologous with the target sequence in the genome of the host cell.Furthermore, the integrational elements may be non-encoding or encodingnucleotide sequences. On the other hand, the vector may be integratedinto the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a polynucleotide of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, llyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E. coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbial. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacterial. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth at al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarisbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth of al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Conolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachietum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Humicola. In a morepreferred aspect, the cell is Humicola insolens. In a most preferredaspect, the cell is Humicola insolens DSM 1800.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell, as described herein, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, wherein the mutant nucleotide sequence encodes a polypeptide thatcomprises or consists of the mature polypeptide of SEQ ID NO: 2; and (b)recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having alpha-L-arabinofuranosidase activity ofthe present invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (several) expression constructs encoding apolypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague ofal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck of al., 1980, Cell 21:285-294, Christensen of al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito of at,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vida faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad of al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chiorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya of al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu of al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu at al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser at al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving alpha-L-arabinofuranosidase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Alpha-L-Arabinofuranosidase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or more(several) nucleotides in the gene or a regulatory element required forthe transcription or translation thereof. For example, nucleotides maybe inserted or removed so as to result in the introduction of a stopcodon, the removal of the start codon, or a change in the open readingframe. Such modification or inactivation may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. Although, in principle, the modificationmay be performed in vivo, i.e., directly on the cell expressing thenucleotide sequence to be modified, it is preferred that themodification be performed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence that is then transformed into the parentcell to produce a defective gene. By homologous recombination, thedefective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of native and/or heterologouspolypeptides. Therefore, the present invention further relates tomethods of producing a native or heterologous polypeptide, comprising:(a) cultivating the mutant cell under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Theterm “heterologous polypeptides” is defined herein as polypeptides thatare not native to the host cell, a native protein in which modificationshave been made to alter the native sequence, or a native protein whoseexpression is quantitatively altered as a result of a manipulation ofthe host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free ofalpha-L-arabinofuranosidase activity by fermentation of a cell thatproduces both a polypeptide of the present invention as well as theprotein product of interest by adding an effective amount of an agentcapable of inhibiting alpha-L-arabinofuranosidase activity to thefermentation broth before, during, or after the fermentation has beencompleted, recovering the product of interest from the fermentationbroth, and optionally subjecting the recovered product to furtherpurification.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free ofalpha-L-arabinofuranosidase activity by cultivating the cell underconditions permitting the expression of the product, subjecting theresultant culture broth to a combined pH and temperature treatment so asto reduce the alpha-L-arabinofuranosidase activity substantially, andrecovering the product from the culture broth. Alternatively, thecombined pH and temperature treatment may be performed on an enzymepreparation recovered from the culture broth. The combined pH andtemperature treatment may optionally be used in combination with atreatment with an alpha-L-arabinofuranosidase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the alpha-L-arabinofuranosidase activity. Complete removal ofalpha-L-arabinofuranosidase activity may be obtained by use of thismethod.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyalpha-L-arabinofuranosidase-free product is of particular interest inthe production of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Thealpha-L-arabinofuranosidase-deficient cells may also be used to expressheterologous proteins of pharmaceutical interest such as hormones,growth factors, receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from alpha-L-arabinofuranosidase activity that isproduced by a method of the present invention.

Methods of Inhibiting Expression of a Polypeptide

The present invention also relates to methods of inhibiting theexpression of a polypeptide in a cell, comprising administering to thecell or expressing in the cell a double-stranded RNA (dsRNA) molecule,wherein the dsRNA comprises a subsequence of a polynucleotide of thepresent invention. In a preferred aspect, the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of a polypeptide in acell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencingtherapeutics. In one aspect, the invention provides methods toselectively degrade RNA using the dsRNAis of the present invention. Theprocess may be practiced in vitro, ex vivo or in vivo. In one aspect,the dsRNA molecules can be used to generate a loss-of-function mutationin a cell, an organ or an animal. Methods for making and using dsRNAmolecules to selectively degrade RNA are well known in the art, see, forexample, U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109; and 6,489,127.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thealpha-L-arabinofuranosidase activity of the composition has beenincreased, e.g., with an enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having alpha-L-arabinofuranosidase activity, orcompositions thereof.

A polypeptide having alpha-L-arabinofuranosidase activity of the presentinvention may be used in several applications to degrade or convert axylan-containing material by treating the material with an effectiveamount of the polypeptide (see, for example, WO 2002/18561). Thepolypeptides of the present invention are preferably used in conjunctionwith other xylan degrading enzymes such as xylanases, acetylxylanesterases, arabinofuranosidases, xylosidases, feruloyl esterases,glucuronidases, and a combination thereof, in processes wherein xylanhas to be degraded. As a consequence of the deacylating reaction thexylan becomes better accessible for xylanases and other xylan-degradingenzymes.

The polypeptides having alpha-L-arabinofuranosidase activity are usefulin a number of applications: in vivo modification of xylan containinganimal feeds to improve digestability; general applications resultingfrom biomass degradation or conversion to fermentable sugars in theproduction of, for example, fuel and/or potable ethanol; processing aidsused in pulp and paper de-lignification; component of enzymatic scouringsystems for textiles; food applications, e.g., baking, in combinationwith other enzymatic functionalities to improve the physical propertiesof baked goods; and laundry detergent applications in combination withother enzyme functionalities.

The polypeptides may be used in methods for the treatment of Kraft pulpaccording to U.S. Pat. No. 5,658,765. Generally Kraft pulp is generallytreated with xylanase in order to remove lignin in the preparation ofpaper products. The effectiveness of xylanase is greatly increased whenpulp is treated with alpha-L-arabinofuranosidase either before or at thesame time as the xylanase treatment.

The polypeptides may also be used in processes for producing xylose orxylo-oligosaccharide according to U.S. Pat. No. 5,658,765.

The polypeptides may also be used as feed enhancing enzymes that improvefeed digestibility to increase the efficiency of its utilizationaccording to U.S. Pat. No. 6,245,546. The use ofalpha-L-arabinofuranosidase in feed can decrease the solubility of thefeed components thereby diminishing the viscosity and reducinganti-nutritional effect of pentosanes.

The polypeptides may also be used in baking according to U.S. Pat. No.5,693,518.

The polypeptides may further be used in brewing according to WO2002/24926, where combinations of this enzyme with other enzymes can beused to degrade biological cell-wall material to increase digestibilityor flow characteristics in applications relating to the preparation offruit juices or beer.

Consequently, the present invention also relates to methods fordegrading a xylan, comprising treating a xylan-containing material withsuch a polypeptide having alpha-L-arabinofuranosidase activity. In apreferred aspect, the xylan-containing material is further treated withone or more xylan degrading enzymes.

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein, wherein the gene is operably linked to anucleotide sequence encoding a signal peptide comprising or consistingof amino acids 1 to 17 of SEQ ID NO: 2, wherein the gene is foreign tothe nucleotide sequence.

In a preferred aspect, the nucleotide sequence comprises or consists ofnucleotides 1 to 51 of SEQ ID NO: 1.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods of producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides that comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more (several) may be heterologous or native to the hostcell. Proteins further include naturally occurring allelic andengineered variations of the above mentioned proteins and hybridproteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Humicola insolens DSM 1800 was used as the source of a Family 62 geneencoding a polypeptide having alpha-L-arabinofuranosidase activity.Aspergillus niger MBin120 strain (WO 2004/090155) was used forexpression of the Humicola insolens gene encoding the polypeptide havingalpha-L-arabinofuranosidase activity.

Media

PDA plates were composed per liter of 39 g of potato dextrose agar.

YP medium was composed per liter of 10 g of yeast extract and 20 g ofBacto peptone.

COVE A urea− acetamide+ plates were composed per liter of 20 ml of COVEA salts solution, 220 g of sorbitol, 10 g of glucose, 10 ml of 1 Macetamide, and 30 g of Bacto agar; pH 5.2.

COVE A salts solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄, 76 g of KH₂PO₄, and 50 ml of COVE A trace elements solution.

COVE A trace elements solution was composed per liter of 0.04 g ofNa₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 0.8 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and 10 g of citricacid.

M410 medium was composed per liter of 50 g of maltose, 50 g of glucose,2 g of MgSO₄.7H₂O, 2 g of KH₂PO₄, 4 g of citric acid anhydrous powder, 8g of yeast extract, 2 g of urea, 0.5 g of AMG trace metals solution, and0.5 g of CaCl₂; pH 6.0.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.7H₂O, and 3 g of citric acid.

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of NaCl.

Example 1 Identification of a Humicola insolens GH62A Polypeptide HavingAlpha-L-Arabinofuranosidase Activity

Protein Fractionation of ULTRAFLO L®. A 2 ml aliquot of ULTRAFLO® L(Novozymes A/S, Bagsvaerd, Denmark) was first buffer-exchanged into 150mM sodium chloride-20 mM sodium acetate pH 5 using a HIPREP™ 26/10Desalting Column (GE Healthcare, Piscataway, N.J., USA). The resultingbuffer-exchanged material (18.5 ml) was then concentrated to 3 ml byultrafiltration with a VIVASPIN® 20 spin column equipped with a 3,000Dalton molecular weight cut-off membrane (Vivascience AG, Hannover,Germany). A 2 ml aliquot of the buffer-exchanged and concentratedULTRAFLO® L material was then fractionated by size-exclusionchromatography over a HILOAD™ 26/60 SUPERDEX™ 200 prep grade sizeexclusion column (GE Healthcare, Piscataway, N.J., USA), under the samebuffer conditions with isocratic elution. Fractions showing UVabsorbance at 280 nm were combined into six separate pools from varyingelution times, ranging from 20-40 ml total volume each. Pooled fractionswere concentrated to between 1-5 ml by ultrafiltration with a VIVASPIN®20 spin column equipped with a 3,000 Da molecular weight cut-offmembrane. Twenty μl of each concentrated pooled fraction was separatedon a CRITERION® 8-16% Tris-HCl SDS-PAGE gel (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) according to the manufacturer's suggestedconditions. PRECISION PLUS PROTEIN™ standards (Bio-Rad Laboratories,Inc., Hercules, Calif., USA) were used as molecular weight markers. Thegel was removed from the cassette and was stained with Coomassie Blue(G250) protein stain (BIO-SAFE™ Coomassie Stain, Bio-Rad Laboratories,Inc., Hercules, Calif., USA), and visible bands were excised with arazor blade for protein identification analysis.

In-gel digestion of polypeptides for peptide sequencing. A MultiPROBE®II Liquid Handling Robot (PerkinElmer Life and Analytical Sciences,Boston, Mass., USA) was used to perform the in-gel digestions. A 45 kDaprotein gel band was reduced with 50 μl of 10 mM dithiothreitol (DTT) in100 mM ammonium bicarbonate pH 8.0 for 30 minutes. Following reduction,the gel piece was alkylated with 50 μl of 55 mM iodoacetamide in 100 mMammonium bicarbonate pH 8.0 for 20 minutes. The dried gel piece wasallowed to swell in 25 μl of a trypsin digestion solution (6 ng/μlsequencing grade trypsin; Promega, Madison, Wis., USA) in 50 mM ammoniumbicarbonate pH 8 for 30 minutes at room temperature, followed by an 8hour digestion at 40° C. Each of the reaction steps described above wasfollowed by numerous washes and pre-washes with the appropriatesolutions following the manufacturer's standard protocol. Fifty μl ofacetonitrile was used to de-hydrate the gel piece between reactions andthe gel piece was air dried between steps. Peptides were extracted twicewith 1% formic acid/2% acetonitrile in HPLC grade water for 30 minutes.Peptide extraction solutions were transferred to a 96 well skirted PCRtype plate (ABGene, Rochester, N.Y., USA) that had been cooled to 10-15°C. and covered with a 96-well plate lid (PerkinElmer Life and AnalyticalSciences, Boston, Mass., USA) to prevent evaporation. Plates werefurther stored at 4° C. until mass spectrometry analysis could beperformed.

Protein Identification. For de novo peptide sequencing by tandem massspectrometry, a Q-TOFMICRO™, a hybrid orthogonal quadrupoletime-of-flight mass spectrometer (Waters Micromass MS Technologies,Milford, Mass.) was used for LC/MS/MS analysis. The Q-TOF MICRO™ wasfitted with an ULTIMATE™ capillary and nano-flow HPLC system, which wascoupled with a FAMOS™ micro autosampler and a SWITCHOS™ II columnswitching device (LCPackings/Dionex, Sunnyvale, Calif., USA) forconcentrating and desalting samples. Samples were loaded onto a guardcolumn (300 μm ID×5 cm, PEPMAP™ C18) fitted in the injection loop andwashed with 0.1% formic acid in water at 40 μl per minute for 2 minutesusing a Switchos II pump. Peptides were separated on a 75 μm ID×15 cm,C18, 3 μm, 100A PEPMAP™ (LC Packings, San Francisco, Calif., USA)nanoflow fused capillary column at a flow rate of 175 nl/minute from asplit flow of 175 μl/minute using a NAN-75 calibrator (Dionex,Sunnyvale, Calif., USA). A step elution gradient of 5% to 80%acetonitrile in 0.1% formic acid was applied over a 45 minute interval.The column eluent was monitored at 215 nm and introduced into the Q-TOFMICRO™ through an electrospray ion source fitted with the nanosprayinterface. The Q-TOF MICRO™ was fully microprocessor controlled usingMASSLYNX™ software version 4.1 (Waters Micromass MS Technologies,Milford, Mass., USA). Data was acquired in survey scan mode and from amass range of m/z 400 to 1990 with the switching criteria for MS toMS/MS to include an ion intensity of greater than 10.0 counts per secondand charge states of +2, +3, and +4. Analysis spectra of up to 4co-eluting species with a scan time of 1.9 seconds and inter-scan timeof 0.1 seconds could be obtained. A cone voltage of 45 volts wastypically used and the collision energy was programmed to be variedaccording to the mass and charge state of the eluting peptide and in therange of 10-60 volts. The acquired spectra were combined, smoothed, andcentered in an automated fashion and a peak list generated. This peaklist was searched against selected databases using PROTEINLYNX™ GlobalServer 2.2.05 software (Waters Micromass MS Technologies, Milford,Mass., USA) and PEAKS Studio version 4.5 SP1 (Bioinformatic SolutionsInc., Waterloo, Ontario, Canada) Results from the PROTEINLYNX™ and PEAKSStudio searches were evaluated and un-identified proteins were analyzedfurther by evaluating the MS/MS spectrums of each ion of interest and denovo sequence was determined by identifying the y and b ion series andmatching mass differences to the appropriate amino acid.

Peptide sequences determined from de novo sequencing by massspectrometry were obtained from several multiply charged ions for thein-gel digested 45 kDa polypeptide gel band. A doubly charged trypticpeptide ion of 60426 m/z sequence was determined to be [[ILE/Leu] orAsp]-Thr-Ser-Glu-Asn-Asn-Pro-Phe-Ala-Gly-Arg (amino acids 249 to 259 ofSEQ ID NO: 2). Another doubly charged tryptic peptide ion of 698.32 m/zsequence was determined to be[Gln/Lys]-Tyr-[Ile/Leu]-Met-[Ile/Leu]-Val-Glu-Ser-[Ile/Leu]-Gly-Ser-Arg(amino acids 218 to 229 of SEQ ID NO: 2). Another doubly charged trypticpeptide ion of 711.32 m/z sequence was determined to beAsn-[Ile/Leu]-Trp-Val-[Ile/Leu]-Ala-Tyr-[Gln/Lys]-Trp-Gly-Arg (aminoacids 105 to 115 of SEQ ID NO: 2). Another doubly charged trypticpeptide ion of 726.84 m/z sequence was determined to beAla-Ala-Val-Ala-Pro-Thr-Leu-Phe-Tyr-Phe-[Gln/Lys]-Pro-Lys (amino acids92 to 104 of SEQ ID NO: 2). Another doubly charged tryptic peptide ionof 1005.42 m/z sequence was determined to beAsn-Asp-[Ile/Leu]-Phe-Glu-Ala-Val-[Gln/Lys]-Val-Tyr-Thr-Ile-Asp-Gly-Ser-Asn-Pro-[Gln/Lys](amino acids 200 to 217 of SEQ ID NO: 2). [Ile/Leu] and [Gln/Lys] couldnot be distinguished because they had equivalent masses.

Example 2 Humicola insolens DSM 1800 Genomic DNA Extraction

Humicola insolens DSM 1800 was grown on PDA plates at 45° C. toconfluence. Three 4 mm² squares were cut from the PDA plates andinoculated into 25 ml of YP medium containing 2% glucose in a baffled125 ml shake flask at 41° C. and 200 rpm for 2 days with shaking at 200rpm. Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem,La Jolla, Calif., USA), washed twice in deionized water, and frozenunder liquid nitrogen. Frozen mycelia were ground, by mortar and pestle,to a fine powder, and total DNA was isolated using a DNEASY® Plant MaxiKit (QIAGEN Inc., Valencia, Calif., USA).

Example 3 Isolation of a Partial Fragment of a GH62AAlpha-L-Arabinofuranosidase Gene from Humicola insolens DSM 1800

Using the Consensus-degenerate hybrid oligonucleotide primer program(CODEHOP; Rose et al., 1998, Nucleic Acids Research 26: 1628-1635),degenerate primers were designed to regions of homology with relatedGH62 sequences based on the identified peptide fragments described inExample 1. Degenerate primers employed to generate a fragment of theHumicola insolens GH62A alpha-L-arabinofuranosidase gene were:

Primer HinsGH62sense1:

5′-CCAAGTCGATCTGGGTNCTCGCNTAYCA-3′ (SEQ ID NO: 3)Protein translation for degenerate primer HinsGH62sense1:

PKSIWVLAYQ (SEQ ID NO: 4)Primer HinsGH62anti1:

5′-AGTTGGCGCGNCCNGCRAANGG-3′ (SEQ ID NO: 5)Protein translation for degenerate primer HinsGH62anti1:

PFAGRAN

To obtain the initial DNA fragment of the Humicola insolens GH62Aalpha-L-arabinofuranosidase gene, gradient PCR was performed at 6different annealing temperatures ranging form 40° C. to 60° C.Amplification reactions (25 μl) were composed of 80 ng of Humicolainsolens DSM 1800 genomic DNA as template, 0.4 mM each of dATP, dTTP,dGTP, and dCTP, 50 pmol each of primer HinsGH62sense1 and primerHinsGH62anti1, 1× ADVANTAGE® GC-Melt LA Buffer (Clontech Laboratories,Inc., Mountain View, Calif., USA), and 1.25 units of ADVANTAGE® GCGenomic Polymerase Mix (Clontech Laboratories, Inc., Mountain View,Calif., USA). The amplification reactions were performed using anEPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury,N.Y., USA) programmed for pre-denaturing at 95° C. for 1 minute; 30cycles each at a denaturing temperature of 95° C. for 30 seconds;annealing temperature of 50° C. +/−10° C. for 30 seconds (6 gradientoptions) and elongation at 72° C. for 1 minute; and final elongation at72° C. for 6 minutes.

The reaction products were isolated by 1.0% agarose gel electrophoresisin TBE (10.8 g of Tris base, 5.5 g of boric acid and 4 ml of 0.5 M EDTApH 8.0 per liter) buffer. A PCR product band of approximately 500 bpfrom an annealing temperature of 59.8° C. was excised from the gel,purified using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia,Calif., USA) according to the manufacturer's instructions, and sequencedwith a Perkin-Elmer Applied Biosystems Model 377 XL Automated DNASequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.,USA) using dye-terminator chemistry (Giesecke et al., 1992, Journal ofVirology Methods 38: 47-60) and primer walking strategy using primersHinsGH62sense1 and Primer HinsGH62anti1.

Example 4 Identification of a Full-Length Humicola insolens GH62AAlpha-L-Arabinofuranosidase Gene

The full-length Family 62 alpha-L-arabinofuranosidase gene wasidentified from Humicola insolens DSM 1800 using a GENOMEWALKER™Universal Kit (Clontech Laboratories, Inc., Mountain View, Calif., USA)according to the manufacturer's instructions. Briefly, total genomic DNAfrom Humicola insolens DSM 1800 was digested separately with fourdifferent restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) thatleave blunt ends. Each batch of digested genomic DNA was then ligatedseparately to the GENOMEWALKER™ Adaptor (Clontech Laboratories, Inc.,Mountain View, Calif., USA) to create four libraries. These fourlibraries were then employed as templates in PCR reactions using fourgene-specific primers shown below, two for a primary and secondary PCRamplifying upstream of the fragment through the 5′ end encoding theN-terminus of the alpha-L-arabinofuranosidase and two for primary andsecondary PCR amplifying downstream of the fragment through the 3′ endencoding the C-terminus of the alpha-L-arabinofuranosidase. Thefollowing primers were designed based on the partial Family 62alpha-L-arabinofuranosidase gene sequence from Humicola insolensdescribed in Example 3.

N-terminus:

Primer Hins_GH62_GSP1_R (primary):

5′-AGCTGTCGCTGATGGAGCCCGAGAAGA-3′ (SEQ ID NO: 6)Primer Hins_GH62_GSP2_R (secondary):

5′-GAAGGCAGTCCGGCCCCATTGATATGC-3′ (SEQ ID NO: 7)C-terminus:Primer Hins_GH62_GSP1_F (primary):

5′-GCTCCAACCCCAAGCAGTACCTCATGC-3′ (SEQ ID NO: 8)Primer Hins_GH62_GSP2_F (secondary):

5′-CCGCTACTTCCGCTCCTACGTCTCCAA-3′ (SEQ ID NO: 9)

The primary amplifications were composed of 1 μl (approximately 6 ng) ofeach library as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10pmol of Adaptor Primer 1 (Clontech Laboratories, Inc., Mountain View,Calif., USA), 50 pmol of primer Hins_GH62_GSP1_R or Hins_GH62_GSP1_F, 1×ADVANTAGE® GC-Melt LA Buffer (Clontech Laboratories, Inc., MountainView, Calif., USA), and 1.25 units of ADVANTAGE® GC Genomic PolymeraseMix in a final volume of 25 μl. The amplifications were performed usingan EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denaturing at 95° C.for 1 minute; 7 cycles each at a denaturing temperature of 95° C. for 25seconds; annealing and elongation at 72° C. for 5 minutes; and 32 cycleseach at a denaturing temperature of 95° C. for 25 seconds; annealing andelongation at 67° C. for 5 minutes; and final elongation at 67° C. for 7minutes.

The secondary amplifications were composed of 1 μl of each primary PCRproduct as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmolof Adaptor Primer 2 (Clontech Laboratories, Inc., Mountain View, Calif.,USA), 50 pmol of primer Hins_GH62_GSP2_R or Hins_GH62_GSP2_F, 1×ADVANTAGE® GC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC GenomicPolymerase Mix in a final volume of 25 μl. The amplifications wereperformed using an EPPENDORF® MASTERCYCLER® 5333 programmed forpre-denaturing at 95° C. for 1 minute; 5 cycles each at a denaturingtemperature of 95° C. for 25 seconds; annealing and elongation at 72° C.for 5 minutes; and 20 cycles each at a denaturing temperature of 95° C.for 25 seconds; annealing and elongation at 67° C. for 7 minutes; andfinal elongation at 67° C. for 5 minutes.

The reaction products were isolated by 1.0% agarose gel electrophoresisin TBE buffer. From the 5′ end PCR amplification, a 600 bp product bandfrom the Dra I library was excised from the gel, purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions, and sequenced. From the 3′ end PCR amplification, a 1.8 kbproduct band from the Dra I library was excised from the gel and a 700bp product band from the Eco RV library was excised from the gel,purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions, and sequenced.

DNA sequencing of the PCR fragments was performed with a Perkin-ElmerApplied Biosystems Model 377 XL Automated DNA Sequencer usingdye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Adaptor Primer 2, primer Hins_GH62_GSP2_R, and primerHins_GH62_GSP2_F were used for sequencing.

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The PCR fragmentsequence results were compared and aligned with the partial Family 62alpha-L-arabinofuranosidase gene sequence from Humicola insolensdescribed in Example 3. A gene model was constructed based on the genefragments obtained here and in Example 3 allowing determination of the5′ and 3′ ends of the gene with other homologous Family 62alpha-L-arabinofuranosidases.

Example 5 Cloning of the Full-Length Humicola insolens GH62AAlpha-L-Arabinofuranosidase Gene and Construction of an Aspergillusniger Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the full-length Humicola insolens DSM 1800alpha-L-arabinofuranosidase gene from the genomic DNA prepared inExample 2. An InFusion Cloning Kit (BD Biosciences, Palo Alto, Calif.,USA) was used to clone the fragment directly into the expression vectorpBM120a (WO 2006/078256).

BDinfGH62senseNCO:

(SEQ ID NO: 10) 5′-ACACAACTGGCCATGAGGTCGGTTGCTGCTTTCCTC-3′BDinfantiGH62PAC1:

(SEQ ID NO: 11) 5′-CAGTCACCTCTAGTTATTACTTACAAGGATTCGAGT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 80 ng of Humicola insolens genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, and 1.25units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of 25μl. The amplification was performed using an EPPENDORF® MASTERCYCLER®5333 programmed for 1 cycle at 94° C. for 1 minute; 5 cycles each at 94°C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 90 seconds; and30 cycles each at 94° C. for 30 seconds, 60° C. for 30 seconds, and 72°C. for 90 seconds; and a final elongation at 72° C. for 5 minutes. Theheat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisin TBE buffer where an approximately 1.1-1.2 kb product band was excisedfrom the gel, and purified using a QIAQUICK® Gel Extraction Kitaccording to the manufacturer's instructions.

Plasmid pBM120a was digested with Nco I and Pac I, isolated by 1.0%agarose gel electrophoresis in TBE buffer, and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

The gene fragment and the digested vector were ligated together using anInFusion Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) resultingin pMMar4 (FIG. 2) in which transcription of thealpha-L-arabinofuranosidase gene was under the control of a hybrid ofpromoters from the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase (NA2-tpi promoter). Theligation reaction (20 μl) was composed of 1× InFusion Buffer (BDBiosciences, Palo Alto, Calif., USA), 1×BSA (BD Biosciences, Palo Alto,Calif., USA), 1 μl of InFusion enzyme (diluted 1:10) (BD Biosciences,Palo Alto, Calif., USA), 106 ng of pBM120a digested with Nco I and PacI, and 163 ng of the purified Humicola insolens PCR product. Thereaction was incubated at room temperature for 30 minutes. Two μl of thereaction was used to transform E. coli XL10 SOLOPACK® GoldSupercompetent cells (Stratagene, La Jolla, Calif., USA). An E. colitransformant containing pMMar4 was detected by restriction digestion andplasmid DNA was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia,Calif., USA). The Humicola insolens GH62A insert in pMMar4 was confirmedby DNA sequencing with a Perkin-Elmer Applied Biosystems Model 377 XLAutomated DNA Sequencer using dye-terminator chemistry (Giesecke et al.,1992, supra) and primer walking strategy. Primers 996271 Na2tpi promoterfwd and 996270 AMG rev, shown below, were used for sequencing.

996271 Na2tpi promoter fwd:

5′-ACTCAATTTACCTCTATCCACACTT-3′ (SEQ. ID NO: 12)996270 AMG rev:

(SEQ. ID NO: 13) 5′-CTATAGCGAAATGGATTGATTGTCT-3′

A clone containing pMMar4 was picked into 2×50 ml of LB mediumsupplemented with 100 μg of ampicillin per ml and grown overnight in 250ml glass beakers at 37° C. and 200 rpm agitation. Plasmid pMMar4 wasisolated from broth using a QIAGEN® Midi Kit according to themanufacturer's instructions. Plasmid pMMar4 was digested with Pme I andisolated by 1.0% agarose gel electrophoresis in TBE buffer, and thefragment containing the GH62A alpha-L-arabinofuranosidase gene waspurified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions in preparation for transforming Aspergillusniger MBin120 protoplasts. The fragment of approximately 1.1-1.2 kb wascloned into pCR®2.1-TOPO® vector (Invitrogen, Carlsbad, Calif., USA)using a TOPO® TA CLONING Kit, to generate pHinsGH62A (FIG. 3). TheHumicola insolens GH62A insert in pHinsGH62A was confirmed by DNAsequencing. E. coli pHinsGH62A was deposited with the AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter, Peoria, Ill., on Nov. 20, 2007.

Example 6 Characterization of the Full-Length Humicola insolens GenomicSequence Encoding a GH62A Polypeptide Having Alpha-L-ArabinofuranosidaseActivity

Nucleotide sequence data (Example 5) were scrutinized for quality andall sequences were compared to each other with assistance of PHRED/PHRAPsoftware (University of Washington, Seattle, Wash., USA).

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) are shown in FIGS. 1A and 1B. The genomic fragmentencodes a polypeptide of 387 amino acids. The % G+C content of thefull-length coding sequence and the mature coding sequence are 64.9% and65%, respectively. Using the SignalP software program (Nielsen at al.,1997, Protein Engineering 10:1-6), a signal peptide of 17 residues waspredicted. The predicted mature protein contains 370 amino acids with amolecular mass of 40.3 kDa.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the mature polypeptide of the Humicola insolens Family 62alpha-L-arabinofuranosidase gene shared 65.7% identity (excluding gaps)to the deduced amino acid sequence of an Aspergillus niger arabinoxylandegrading enzyme (GeneSeqP accession number AAR94170).

Example 7 Expression of the Humicola Insolens GH62AAlpha-L-Arabinofuranosidase Gene in Aspergillus niger MBin120

Aspergillus niger MBin120 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Four μgof Pme I digested pMMar4 were used to transform Aspergillus nigerMBin120.

The transformation of Aspergillus niger MBin120 with the Pme I digestedpMMar4 yielded about 50 transformants. Twenty-two transformants wereisolated to individual COVE A urea− acetamide+ plates. Two 3 mm squareagar plugs were cut from confluent COVE A urea-acetamide+plates of the22 transformants and inoculated separately into 25 ml of M410 medium in125 ml plastic shake flasks and incubated at 34° C., 250 rpm. After 5days incubation, 6 μl of supernatant from each culture were analyzed bySDS-PAGE using a CRITERION® 8-16% Tris-HCl SOS-PAGE gel with aCRITERION® Cell (Bio-Rad Laboratories, Inc., Hercules, Calif., USA),according to the manufacturer's instructions. The resulting gel wasstained with 810-SAFE™ Coomassie Stain (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA). SDS-PAGE profiles of the cultures showedapproximately half of the transformants had a major band ofapproximately 45 kDa. One transformant designated Aspergillus nigerMMar202 was chosen for expression of the Humicola insolens GH62Apolypeptide having alpha-L-arabinofuranosidase activity in Aspergillusniger.

Example 8 Fermentation of Aspergillus niger MMar202

Shake flask medium was composed per liter of 70 g of sucrose and 100 gof soy concentrate. Trace metals solution was composed per liter of 13.8g of FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 11.6 g of MnSO₄.H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O and 3.3 g of citric acid monohydrate.

One hundred ml of shake flask medium was added to a 500 ml shake flask.The shake flask was inoculated with 200 μl from a glycerol spore stockof Aspergillus niger MMar202 and incubated at 30° C. on an orbitalshaker at 220 rpm for 72 hours. Fifty ml of the shake flask broth fromeach of four separate shake flasks was used to inoculate a 3 literfermentation vessel.

Fermentation batch medium was composed per liter of 250 g of glucose, 5g of (NH₄)₂SO₄, 2.5 g of KH₂PO₄, 0.5 g of CaCl₂.2H₂O, 2 g of MgSO₄.7H₂O,3 g of K₂SO₄, 1 g of citric acid, 1 ml of anti-foam, and 0.75 ml oftrace metals solution. The trace metals solution was composed per literof 13.8 g of FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 11.6 g of MnSO₄.H₂O, 2.5g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, and 3.3 g of citric acidmonohydrate. Fermentation feed medium was composed per kilogram of 406 gof maltose, 0.5 g of citric acid monohydrate, and 1 ml of anti-foam.

A total of 2 liters of the fermentation batch medium was added to anApplikon Biotechnology two liter glass jacketed fermentor (ApplikonBiotechnology, Schiedam, Netherlands). Fermentation feed medium wasdosed at a rate of 0 to 4 g/l/hr for a period of 185 hours. Thefermentation vessel was maintained at a temperature of 34° C. and pH wascontrolled using an Applikon 1030 control system (ApplikonBiotechnology, Schiedam, Netherlands) to a set-point of 5.1+/−0.1. Airwas added to the vessel at a rate of 1 vvm and the broth was agitated byRushton impeller rotating at 1100 rpm. At the end of the fermentation,whole broth was harvested from the vessel and centrifuged at 3000×g. toremove the biomass. The supernatant was sterile filtered and stored at 5to 10° C.

Example 9 Purification of Humicola insolens GH62A Polypeptide HavingAlpha-L-Arabinofuranosidase Activity

Fermentation broth supernatant (Example 8) containing recombinantHumicola insolens GH62A polypeptide having alpha-L-arabinofuranosidaseactivity expressed in Aspergillus niger was first buffer-exchanged into25 mM sodium acetate pH 5.1 by passing through 400 ml of SEPHADEX™ G-25fine resin (GE Healthcare, Piscataway, N.J., USA) equilibrated in thesame buffer. The resulting buffer-exchanged material (50 ml) was thenpurified using a MONO S™ HR 16/10 column (GE Healthcare, Piscataway,N.J., USA) equilibrated with the same buffer, and then eluted with alinear gradient of 0-0.5 M sodium chloride. Next, fractions showing UVabsorbance at 280 nm were analyzed by SDS-PAGE. A 2.5 μl fractionaliquot was separated on a CRITERION® 8-16% Tris-HCl SDS-PAGE gelaccording to the manufacturer's suggested conditions. PRECISION PLUSPROTEIN™ standards were used as molecular weight markers. The gel wasremoved from the cassette and stained with INSTANTBLUE™ Coomassie Blueprotein stain (Expedeon Protein Solutions, Cambridge, UK) according tothe manufacturer's suggested conditions. Fractions showing UV absorbanceat 280 nm were also assayed for activity with medium viscosity wheatarabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow,Ireland). Fractions were diluted and incubated in a 96-well COSTAR®microtiter plate (Corning Inc., Corning, N.Y., USA) with 4.75 mg ofwheat arabinoxylan per ml of 50 mM sodium acetate pH 5 with 0.01% (w/v)TWEEN® 20 in a total volume of 200 μl for 70 minutes at 40° C. Thereaction was stopped by addition of 50 μl of 2% sodium hydroxide, andthe reducing sugar content determined using a para-hydroxybenzoic acidhydrazide (PHBAH, Sigma, St. Louis, Mo., USA) assay adapted to a 96 wellmicroplate format as described below. Briefly, a 100 μl aliquot ofsample was placed in a 96 well conical bottom COSTAR® microtiter plate.Reactions were initiated by adding 50 μl of 1.5% (w/v) PHBAH in 2% NaOHto each well. Plates were heated uncovered at 95° C. for 10 minutes.Plates were allowed to cool to room temperature and 50 μl of H₂O addedto each well. A 100 μl aliquot from each well was transferred to a flatbottom 96 well plate and the absorbance at 410 nm was measured using aSPECTRAMAX® Microplate Reader (Molecular Devices, Sunnyvale, Calif.,USA). Glucose standards (0.100-0.065 mg/ml) were used to prepare astandard curve to convert the obtained absorbance values at 410 nm intoglucose equivalents, and quantify the amount of reducing sugars releasedin the assay. Fractions containing a long, diffuse band on SDS-PAGE from40-150 kD, and also having activity with wheat arabinoxylan, werepooled, with a total volume of 48 ml.

The pooled material was next concentrated to a 1.5 ml volume using aVIVASPIN™ 20 ultrafiltration concentrator with a 10 kDa molecular weightcut-off membrane. The concentrated material was then purified using aHILOAD™ 26/60 SUPERDEX™ 75 Prep Grade column (GE Healthcare, Piscataway,N.J., USA) in 25 mM sodium acetate pH 5 with 125 mM sodium chloride.Column fractions were analyzed and pooled in a similar manner bySDS-PAGE and wheat arabinoxylan activity as described above, to yieldpurified Humicola insolens alpha-L-arabinofuranosidase. Proteinconcentration of the purified Humicola insolensalpha-L-arabinofuranosidase was determined using a Microplate BCA™Protein Assay Kit (Pierce, Rockford, Ill., USA).

Example 10 Enzyme Activity of the Humicola insolens GH62A PolypeptideHaving Alpha-L-Arabinofuranosidase Activity

Purified Humicola insolens GH62A polypeptide havingalpha-L-arabinofuranosidase activity (Example 9) was diluted andincubated in a 96-well COSTAR® microtiter plate with 5 mg of mediumviscosity wheat arabinoxylan (Megazyme International Ireland, Ltd.,Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in atotal volume of 200 μl for 30 minutes at 40° C. After incubation, theplate was cooled on ice, and then the reactions were filtered throughULTRAFREE®-0.5 centrifugal filters with BIOMAX® 5 kDa molecular weightcut-off membranes (Millipore, Billerica, Mass., USA). The filtrates werethen analyzed for sugar content.

Sugar concentrations of sample filtrates were measured after elution by0.005 M sulfuric acid with 0.05% w/w benzoic acid at a flow rate of 0.6ml per minute from a 4.6×250 mm AMINEX® HPX-87H column (Bio-RadLaboratories, Inc., Hercules, Calif., USA) at 65° C. with quantitationby integration of glucose, arabinose, and xylose signals from refractiveindex detector (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies,Santa Clara, Calif., USA) calibrated by pure sugar samples. The Humicolainsolens polypeptide having alpha-L-arabinofuranosidase activity wasobserved to release 92.6 mg of arabinose/mg enzyme protein and 7.4 mg ofxylose/mg enzyme protein from wheat arabinoxylan under the assayconditions.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, USA, and given the followingaccession number:

Deposit Accession Number Date of Deposit E. coli pHinsGH62A NRRL B-50075Nov. 20, 2007

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. An isolated polypeptide having alpha-L-arabinofuranosidase activity,selected from the group consisting of: (a) a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the maturepolypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least high stringency conditionswith the mature polypeptide coding sequence of SEQ ID NO: 1 or itsfull-length complementary strand, wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide, and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; and (c) a polypeptide encoded by a polynucleotidecomprising a nucleotide sequence having at least 95% sequence identityto the mature polypeptide coding sequence of SEQ ID NO:
 1. 2. Thepolypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:2; or a fragment thereof having alpha-L-arabinofuranosidase activity. 3.The polypeptide of claim 1, which is encoded by a polynucleotidecomprising or consisting of the nucleotide sequence of SEQ ID NO: 1; ora subsequence thereof encoding a fragment havingalpha-L-arabinofuranosidase activity.
 4. The polypeptide of claim 1,which is encoded by the polynucleotide contained in plasmid pHinsGH62Awhich is contained in E. coli NRRL B-50075.
 5. A method of producing thepolypeptide of claim 1, comprising: (a) cultivating a cell, which in itswild-type form produces the polypeptide, under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 6. Amethod for degrading a xylan, comprising treating a xylan-containingmaterial with the polypeptide having alpha-L-arabinofuranosidaseactivity of claim
 1. 7. The method of claim 6, further comprisingtreating the xylan-containing material with one or more (several) xylandegrading enzymes.
 8. The polypeptide of claim 1, comprising an aminoacid sequence having at least 97% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 9. The polypeptide of claim 1, which isencoded by a polynucleotide that hybridizes under at least very highstringency conditions with the mature polypeptide coding sequence of SEQID NO: 1 or its full-length complementary strand, wherein very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 50% formamide, and washing three times each for 15minutes using 2×SSC, 0.2% SDS at 70° C.
 10. The polypeptide of claim 1,which is encoded by a polynucleotide comprising a nucleotide sequencehaving at least 97% sequence identity to the mature polypeptide codingsequence of SEQ ID NO:
 1. 11. The polypeptide of claim 1, comprising themature polypeptide of SEQ ID NO:
 2. 12. The polypeptide of claim 1,consisting of the amino acid sequence of SEQ ID NO: 2; or a fragmentthereof having alpha-L-arabinofuranosidase activity.
 13. The polypeptideof claim 12, consisting of the amino acid sequence of SEQ ID NO:
 2. 14.The polypeptide of claim 1, consisting of the mature polypeptide of SEQID NO: 2.